PROJECT SUMMARY The NADPH oxidase DUOX1 is prominently expressed within the respiratory epithelium and contributes to innate response mechanisms to injury or other environmental triggers, by generating reactive oxygen species (ROS) and activating redox-dependent signaling pathways. In the present funding cycle of this project, we identified DUOX1 as a critical mediator of innate epithelial responses to allergens, by promoting epithelial secretion of the alarmin IL-33 and subsequent activation of type 2 inflammation. We also observed enhanced epithelial DUOX1 in subjects with allergic asthma, and a critical role for DUOX1 in development of various critical features of house dust mite (HDM)-induced allergic airway inflammation in mice, such as mucus metaplasia, subepithelial fibrosis, and airway hyperresponsiveness. These actions of DUOX1 were largely related to redox-dependent activation of Src family kinases and epidermal growth factor receptor (EGFR) signaling, both well-recognized factors in allergic inflammation and airway remodeling, in part by mediating cysteine oxidation within these kinases. During these studies, we noted that DUOX1 is not only operative within the airway epithelium, but is also present in non- epithelial cell types such as alveolar macrophages, and appears to be involved in macrophage polarization and IL-33-mediated pro-fibrotic mediators such as IL-13 and TGF-? that are involved in airway remodeling. The first aim of this renewal application is to delineate the cell-specific actions of DUOX1 on various aspects of HDM- induced allergic inflammation and remodeling, specifically focusing on a potential role for DUOX1 in alternative neutrophil (N2) polarization or macrophage (M2) activation. Aim 2 is to identify redox-sensitive targets of DUOX1 and characterize the molecular mechanisms by DUOX1-dependent cysteine oxidation regulates the enzymatic function of Src, using molecular dynamics simulations and experimental studies with various cysteine variants. Finally, based on previous findings that thiol-reactive electrophiles can inhibit DUOX1 activation, aim 3 will be to develop peptide-based cysteine-targeted approaches to pharmacologically inhibit DUOX1 activity, thus filling an unmet need for DUOX1-selective inhibitors that may be used for clinical development in treatment of severe asthma.